Anaplasma phagocytophilum and Ehrlichia chaffeensis cause emerging potentially fatal infectious diseases human granulocytic anaplasmosis (HGA) and human monocytic ehrlichiosis (HME), respectively. These pathogens are fastidious obligatory intracellular bacteria that infect human leukocytes, and ticks are biological vectors for transmission. How these bacteria enter and continue to thrive within hostile host milieu such as neutrophils, macrophages, and the tick cells is largely unknown. Our hypothesis is bacterial Type IV secretion (T4S) system and two-component system (TCS) play important roles in this process. The specific aims of this project are as follows: 1. Characterize T4S effector molecules, interacting host proteins, and downstream events using yeast two-hybrid system, co-immunoprecipitation, double immunofluorescence labeling, and various pharmacological signal inhibitors and inducers, and by phenotype analysis of host cells transfected with wild-typeT4S effectors or mutant effectors with appropriate modifications (truncation, amino acid substitution, etc.). 2. Characterize CtrA function by analyzing the temporal expression of CckA and CtrA during intracellular replication and development of E. chaffeensis and A. phagocytophilum in human leukocytes and tick cells, and demonstrating CtrA regulation of genes with the upstream CtrA consensus binding site. 3. Identify genes regulated by NtrX by affinity purification of genomic DNA fragments bound to the activated recombinant NtrX, sequencing the DNA fragments, and investigate the downstream events. 4. Characterize PleD function of E. chaffeensis and A. phagocytophilum by analyzing the PleC and PleD temporal expression in human leukocytes and tick cells, verifying PleD diguanyl cyclase activity and identifying c-di-GMP binding protein targets and their functions. The data to be obtained from this study will continue to provide a breakthrough to new understanding of the dynamic signaling events between obligatory intracellular bacteria and their hosts. The results may point to a potential target for treatment and prevention of HME and HGA.